Lipid transfer proteins are important regulators of cellular processes that couple lipid metabolism with cellular signaling events. The long term goal of the Yao lab is to a reach a general understanding of the fundamental biophysical and biochemical mechanisms of lipid transfer protein functions. In this proposal, we seek to dissect the structural and biochemical basis of the functional regulation of the ceramide transfer protein (CERT). CERT mediates non-vesicular transport of ceramide from ER to Golgi for sphingomyelin synthesis. This generates diacylglycerol, which itself is a critical regulator of vesicular trafficking in the Golgi. In this manner, CERT couples sphingolipid metabolism with the regulation of Golgi trafficking. CERT contains multiple domains and motifs. Its N-terminal pleckstrin homology (PH) domain binds to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane and targets CERT to the Golgi. The ceramide transfer activity of CERT is carried out by the C-terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. Hyper-phosphorylation of a serine rich (SR) motif after the PH domain decreases its activity. Recent reports suggest that the PH and START domains physically interact in phosphorylated CERT and that this interaction is responsible for inhibition of both PH binding to PtdIns(4)P and START domain transfer of ceramide to the Golgi. Significantly, our preliminary studies show that the middle region (MR) of CERT is required for both CERT binding to PtdIns(4)P and phosphorylation inhibition of ceramide transfer activity. MR contains no obvious domain(s), however similarly regions are also found in a number of other lipid transfer proteins. This suggests that these regions fulfill an evolutionarily conserved, but as yet unappreciated role in regulating lipid transfer protein function. Our preliminary data show isolated MR binds the START domain. These observations lead to the following hypothesis. In unphosphorylated CERT, the START domain engages in intramolecular interaction with MR, thus preventing START association with PH. Phosphorylation switches START domain to interact with the PH domain and leads to inhibition of CERT activity. We will use a combination of x-ray crystallography, nuclear magnetic resonance and biochemical tools to test this central hypothesis. CERT function is essential for development, anti-oxidative response and normal life span and has implications in a variety of diseases such as cancer, aging, virus and bacteria infections. In-depth dissection of CERT functional regulation would allow the development of novel tools to manipulate CERT function for both research and therapeutic purposes. Abstract